The present invention relates to amino acid sequences that are directed against (as defined herein) chemokines, as well as to compounds or constructs, and in particular proteins and polypeptides, that comprise or essentially consist of one or more such amino acid sequences (also referred to herein as “amino acid sequences of the invention”, “compounds of the invention”, and “polypeptides of the invention”, respectively).
The invention also relates to nucleic acids encoding such amino acid sequences and polypeptides (also referred to herein as “nucleic acids of the invention” or “nucleotide sequences of the invention”); to methods for preparing such amino acid sequences and polypeptides; to host cells expressing or capable of expressing such amino acid sequences or polypeptides; to compositions, and in particular to pharmaceutical compositions, that comprise such amino acid sequences, polypeptides, nucleic acids and/or host cells; and to uses of such amino acid sequences or polypeptides, nucleic acids, host cells and/or compositions, in particular for prophylactic, therapeutic or diagnostic purposes, such as the prophylactic, therapeutic or diagnostic purposes mentioned herein.
Other aspects, embodiments, advantages and applications of the invention will, become clear from the further description herein.
Chemokines (or chemotactic cytokines) are a large family of small soluble proteins which, despite their low sequence homology, exhibit a common basic structural fold known as the “chemokine fold”. This structure consists of a short N-terminal region, an extended N-loop followed by three beta-strands and an alpha-helix. Chemokines are further classified based on the presence of conserved cysteine residues near the N-terminus, resulting in the different subfamilies: “CCL” (or “CC”), “CXCL” (or “CXC”), “XCL” (or “XC”) and “CX3CL” (or “CX3C”) (see review Rajagopalan L. and Rajarathnam K. Biosci. Rep. (2006) 26(5): 325-339). The CC family of chemokines and chemokine receptors and the CXC, CX3C and XC families of chemokines and chemokine receptors are shown in FIGS. 1 and 2 respectively. For a further description of chemokines, their subfamilies, their biological functions and diseases and disorders with which they are associated, reference is inter alia made to the following reviews: Wells et al. Trends in Pharmacological Sciences (2006) 27(1): 41-47; Stein et al. Immunology (2005) 116 (1): 1-12; Bendall., Histol. Histopathol. (2005) 20: 907-926); Rajagopalan & Rajarathnam, Biosci Rep 26, 325-39 (2006); Weber et al., (2006); Trends Immunol, (27), 6, 268-73; and Charo et al., (2006), N Engl J Med, (354), 6, 610-21.
Chemokines belonging to these different subfamilies are well known in the art (see again the reviews by Wells et al. and by Stein et al.) and include but are not limited to CCL1/I-309, CCL2/MCP-1, CCL3/MIP-1-alpha, CCL3L1/LD-78-beta, CCL4/MIP-1-beta, CCL5/RANTES, CCL6/C10, CCL7/MCP-3, CCL8/MCP-2, CCL9/10/MIP-1-gamma, CCL11/Eotaxin, CCL12/MCP-5, CCL13/MCP-4, CCL14/HCC-1, CCL15/HCC-2, CCL16/HCC-4, CCL17/TARC, CCL18/PARC, CCL19/ELC, CCL20/MIP-3-alpha, CCL21/SLC, CCL22/MDC, CCL23/MPIF-1, CCL24/Eotaxin-2, CCL25/TECK, CCL26/Eotaxin-3, CCL27/CTACK, CCL28/MEC, CXCL1/Gro-alpha, CXCL2/Gro-beta CXCL3/Gro-gamma, CXCL4/PF4, CXCL5/ENA-78, CXCL6/GCP-2, CXCL7/NAP-2, CXCL8/IL-8, CXCL9/Mig, CXCL10/IP-10, CXCL11/I-TAC, CXCL12/SDF-1-alpha/beta, CXCL13/BCA-1, CXCL14/BRAK, CXCL15/Lungkine, CXCL16, XCL1/Lymphotactin, XCL2/SCM-1-beta and CX3CL1/Fractalkine.
Chemokines are prominent players of inflammation and immunity: neutralizing chemokines has proven to relief the inflammatory and immune response in a large spectrum of diseases, offers considerable benefits in terms of specificity and side effects, and enables the design of more effective medicines to treat a wide range of inflammatory diseases, immune diseases and cancer. Chemokine receptors, their ligands and their association to disease is illustrated in FIG. 3.
Chemokines interact with glycosaminoglycans (GAGS) (Hoogewerf et al. Biochemistry (1997) 36 (44): 13570-13578; Kuschert et al. Biochemistry (1999) 38 (39): 12959-12968; Lau et al. J. Biol. Chem. (2004) 279 (21): 22294-22305; Murooka et al. J. Biol. Chem. (2006) 281 (35): 25184-25194; Shaw et al. Structure (2004) 12 (11): 2081-2093). This interaction has been long suggested to be a requisite for the chemokine gradients that build up at inflammatory sites, and which allow direct migration of specific cell types (Wells et al. Trends in Pharmacological Sciences (2006) 27(1): 41-47). Molecules that break this chemokine gradient have been shown to be anti-inflammatory agents. Chemokines oligomerize on GAG surfaces where both GAG binding and higher-order quaternary structure are essential for their activity in vivo (Wells et al. Trends in Pharmacological. Sciences (2006) 27(1): 41-47; Proudfoot PNAS (2003) 100(4): 1885-1890; Weber et al. Trends in Immunology (2006) 27(6): 268-273).
The chemokine CCL2 is involved in the development of atherosclerosis, rheumatoid arthritis (RA), lung inflammatory diseases (including asthma), multiple sclerosis (MS), organ transplant rejection, and peripheral neuropathy. In vascular diseases, like atherosclerosis, which is a chronic inflammatory disease, CCL2 is produced by endothelial cells and promotes the development of this disease. This was initially proven using the CCL2−/− or CCR2−/− mouse model, which showed that mutant mice were more resistant to atherosclerosis. Moreover, genomic studies showed that a polymorphism in the CCL2 promoter is associated with increased transcription of CCL2 and a higher risk of coronary artery disease (see review Charo et al. New England Journal of Medicine (2006) 354 (6): 610-621 and references therein). In obesity-induced diabetes, CCR2 is expressed by adipocytes, which, when activated by CCL2, causes expression of inflammatory genes and impaired uptake of insulin-dependent glucose. It was indeed shown that CCR2−/− mice have improved insulin resistance (see review Charo et al. New England Journal of Medicine (2006) 354(6): 610-621 and references therein). In cancer, tumours secrete pro-inflammatory chemokines and cytokines, which recruit leukocytes that, upon activation, release angiogenic factors, mitogens, proteolytic enzymes and other chemotactic factors, which in their turn recruit more inflammatory cells and sustain tumour growth, invasion and angiogenesis (see review Yan et al. European Journal of Cancer (2006) 42: 793-802 and references therein). CCL2 was found to be a prognostic biomarker indicating poor prognosis and early relapse. High levels of CCL2 expression in tumours has been correlated to tumour associated macrophage infiltration in many cancers (see review Yan et al. European Journal of Cancer (2006) 42: 793-802 and references therein). CCL2 is a potent pro-angiogenic factor and neutralizing antibodies were able to block in viva tumour angiogenesis (see review Yan et al. European Journal of Cancer (2006) 42: 793-802 and references therein).
The chemokines CCL3 and CCL5 are known to be involved in the development of MS, RA, organ transplant rejection, allergy, asthma, nephritis, inflammatory bowel disease, and AIDS. In rheumatoid arthritis. CCL2, CCL3 and CCL5 have particularly elevated levels in the joints of patients. In immune diseases like multiple sclerosis, chemokines like CCL5 and CXCL10 play multiple roles that will influence the progression and severity of the disease by attracting immune cells to lesion sites (see review Wells et al. Trends in Pharmacological Sciences (2006) 27(1): 41-47 and references therein). In asthma, also CCL11 and its receptor CCR3 play an important role by contributing to the recruitment of eosinophils in the lung.
CXCL8 neutralizing antibody (Abgenix) has proven to be useful in the treatment of psoriasis, melanoma and chronic obstructive pulmonary disease (COPD) in animals models (see review Wells et al. Trends in Pharmacological Sciences (2006) 27(1): 41-47 and references therein). As already mentioned above, some chemokines have pro-angiogenic properties. Therefore, neutralizing the chemokine will prevent cancer-related angiogenesis and tumour progression (see review Stricter et al. Cytokine and Growth factor Reviews (2005) 16: 593-609; Kakinuma et al. J. Leukoc. Biol. (2006) 79 (4): 639-651). CXCL8 maintains the angiogenic phenotype of the endothelial cell. In an immunodeficient mouse model of NSCLC, depletion of CXCL5 and CXCL8 inhibited the enhanced tumour growth of COX2 overexpressing tumours. Inhibition of CXCL8 markedly reduced the tumour growth and tumour associated angiogenesis of glioblastomas expressing low levels of ING4 (see review Stricter et al. Cytokine and Growth factor Reviews (2005) 16: 593-609).
Also CXCL11 plays a crucial role in MS, RA, and organ transplant rejection (Kao et al. Circulation (2003) 107 (15): 1958-1961; Burns et al. J. Exp. Med. (2006) 203 (9): 2201-2213) whereas CXCL12 is involved in wound healing, AIDS and cancer. CXCL11 is angiostatic, and its neutralization could have great effect on diseases that require angiogenesis for treatment. In transplant rejection, the infiltration of leukocytes driven by chemokines into an allograft is an essential component of organ transplant rejection. CXCR3 activation by CXCL9, CXCL10 or CXCL11 has been involved in many allograft rejections. CXCR3-deficient recipients and recipients of grafts from CXCL10-deficient donors both experience prolonged graft survival (see review Tan et al. Cellular & Molecular Immunology (2005)).
In psoriasis, infiltrating effector T cells express CCL17, CCL22 and their receptor CCR4 (see review Charo et al. New England Journal of Medicine (2006) 354 (6): 610-621 and references therein). Neutralizing the chemokine will prevent monocyte and T cells recruitment and may relieve the symptoms. In pain, upregulation of chemokines resulting from macrophage infiltration at the site of nerve damage, is one of the mechanisms promoting the development and establishment of several types of pain (see reviews Abbadie et al. PNAS (2003) 100 (13): 7947-7952; Abbadie, Trends in Immunology (2005) 26(10): 529-534).